Telemore length Analysis using RT-PCR: We have implemented a real-time quantitative PCR assay for measuring telomere length. Recent molecular epidemiologic studies of telomere length in human peripheral blood have provided evidence that telomere length is inversely related to age and many age-related disorders including insulin resistance, atherosclerosis, cardiovascular disease, Alzheimers disease, infectious disease and cancer. In addition there is evidence that telomere length is associated with obesity and smoking and with inflammation and estrogen, probably via oxidative stress.[unreadable] [unreadable] We are implementing this assay in two large molecular epidemiology studies. The first is of telomere length in relation to cancer in the Sister Study, starting with an initial evaluation of 350 breast cancer cases and 700 controls. The second is to study the genetics of telomere length as a quantitative trait, using 500 mother-father-child trios from Norway on whom we will soon have 500,000 single nucleotide polymorphism (SNP) data from a genome wide association study that is being completed on these sample this summer.[unreadable] [unreadable] Telomere Length Analysis using Quantitative Fluorescence In-Situ Hybridization (Q-FISH): We have also developed the Q-FISH assay for our group using cultured human lymphoblastoid cell lines harboring telomeres of known length as standards. Mean telomere length of individual interphase cells is determined using fluorescently labeled peptide nucleic acid (PNA) probes complementary to telomere repeats. The superior annealing properties of PNA probes compared to DNA probes enables quantitative comparison of mean telomere length of one population of cells compared to another based on fluorescent signal intensities. The assay takes advantage of our automated epifluorescent microscope system that can load slides onto the stage, search for and focus on individual cells, then capture and analyze digital images according to a user defined processing algorithm. [unreadable] With the Q-FISH assay verified using cultured cells, we are now adapting the assay to measure telomere length of cells in paraffin-embedded, formalin-fixed tissue sections from human tumor samples. This type of analysis will allow in-situ comparison of telomere length in tumor cells with that of adjacent normal cells, and will help us understand the processes of telomere attrition and maintenance during carcinogenesis. This assay will be implemented in tissue microarrays of prostate cancer.[unreadable] [unreadable] [unreadable] [unreadable] DNA Methylation: We undertaking two large scale molecular epidemiologic studies of global methylation. These studies address several hypotheses: that global methylation levels in blood DNA are predictors for disease (specifically breast cancer and cleft lip/palate), that environmental exposure is associated with methylation levels, and that inheritance of selected polymorphisms controls global methylation as a quantitative trait. To do these studies we are using pyrosequencing, which provides a very precise measure of methylation that can efficiently be applied to large numbers of samples for small numbers of specific CpG sites. During the past year we have perfected pyrosequencing methods for measuring gene-specific and global methylation in blood samples. Based on this success we are extending this work in a nested case-control study of breast cancer within the NIEHS Sister Study. The hypothesis is to test for an association between peripheral blood DNA methylation of both genome-wide repetitive elements and gene-specific promoter hypermethylation and breast cancer risk in women. We have identified several candidate genes which undergo methylation in breast cancer, designed and optimized pyrosequencing assays, and have acquired preliminary methylation data using a panel of healthy donors. We developed a study proposal which was presented to the Epidemiology Branch and approved. In addition, a proposal for supporting work of DNA extraction and genotyping for SNPs involved in single carbon metabolism was approved by the Molecular Genetics Core Facility Advisory Committee. In addition we have done a pilot work using an Illumina methylation array in collaboration with Dr. Bells group to identify novel methylation targets which will then be added to the current gene-specific methylation panel to assess risk in the Sister Study samples